Mammals acquire cholesterol by two pathways: de novo synthesis of cholesterol and cholesterol absorption. The contribution of each of these pathways in humans is roughly equivalent, and in the last several years, with the identification of proteins integral to cholesterol absorption, there is increased interest in identifying ways to regulate intestinal sterol uptake to ultimately reduce serum LDL-cholesterol levels. In previous work, we observed that the retinoid X receptor (RXR) ligand LG268 dramatically reduced cholesterol absorption in mice. Further studies revealed that this activity could be attributed to RXR's role as an obligate heterodimer partner with two nuclear orphan receptors: the liver X receptor (LXR) by regulation of ATP-binding cassette transporters ABCG5/G8;and the farnesoid X receptor (FXR) by regulation of bile acid production. In preliminary work presented here, we establish that LG268 continues to affect cholesterol absorption in a manner independent of LXR and FXR. Therefore, the long-term objective of this proposal is to elucidate the molecular mechanism(s) responsible for RXR-mediated inhibition of cholesterol absorption and identify additional means of regulating this important cholesterol acquisition pathway. Specific Aim 1. Define the molecular mechanism of action of RXR. We will use various nuclear hormone receptor knockout mice, an intestinal explant culture system, and selective synthetic ligands to establish if RXR is acting as a homodimer, an obligate heterodimer partner, or through interaction with another transcription factor (transrepression) to reduce cholesterol absorption. Specific Aim 2. Identify the ultimate intestinal target gene responsible for RXR-mediated inhibition of cholesterol absorption. Of all the genes evaluated thus far, only NPC1L1, a putative cholesterol transporter shows altered expression by RXR. We will use the human Caco2 cell line to interrogate the NPC1L1 promoter by cell reporter assay. Specific Aim 3. Identify the RXR-regulated primary target gene that affects NPC1L1 expression. Preliminary results establish that RXR does not act directly on the NPC1L1 gene, but induces production of an intermediate responsible for NPC1L1 transcriptional regulation. We will use microarray, ChIP-on-Chip, and NPC1L1-promoter ChIP methods to define the transcriptional regulators of NPC1L1. PUBLICH HEALTH RELEVANCE: The studies proposed in this application will reveal novel targets for cholesterol-lowering therapies to control serum LDL-cholesterol levels. Additional mechanisms to reduce the cholesterol transport protein, NPC1L1, particularly in an intestine-specific manner, may result in the development of safer drugs for the treatment of hypercholesterolemia and cholelithiasis.